Vitreoscilla is a filamentous myxobacterium with a simple respiratory chain for which cytochrome-o serves as the terminal oxidase. There are no a-, c-, or d-type cytochromes present in this organism. Two components of its NADH-cytochrome o oxidase system have been solubilized and purified: cytochrome-o and NADH-cytochrome-o reductase. The cytochrome has a MW of 27,000 with two identical 13,000 MW subunits and two hemes per molecule. Projected studies on this protein include anaerobic potentiometric titrations to determine its electron-accepting potentials and analysis of its reaction with oxygen using rapid kinetic techniques (stopped-flow). The NADH-cytochrome-o reductase is a protein of 61,000 MW containing one FAD per molecule. The NADH-cytochrome-o oxidase system contains additional components, which have not yet been separated. Once this system has been reconstituted in vitro the reaction of the reductase with cytochrome-o will be studied and the sites of action of specific inhibitors determined. Results obtained with the soluble system will be used to investigate the physiological function of each component in respiratory membrane fragments (RMFs). This will enable interpretation of the action of specific inhibitors on the RMFs and a study of hydrogen peroxide generation by these fragments. The RMFs can also be used as a starting point for obtaining a soluble oxidative phosphorylating system from these bacteria. BIBLIOGRAPHIC REFERENCES: Webster, D.A. and Y. Orii (1977) Oxygenated Cytochrome o. An active intermediate observed in whole cells of Vitreoscilla. J. Biol. Chem., in press. Orii, Y. and D.A. Webster (1977) Formation of oxygentated Cytochrome o (Vitreoscilla) by treating the oxidized cytochrome with superoxide anion. Plant Cell Physiol. in press.